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Results for “Chain Migration”

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PubMed1988

Newt epidermal cell migration over collagen and fibronectin involves different mechanisms.

Effects of the synthetic peptides, Arg-Gly-Asp-Ser (RGDS), the amino acid sequence representing the fibroblast attachment site in fibronectin, and Arg-Gly-Glu-Ser (RGES), on collagen- and fibronectin-mediated migration in newt epidermal cells were compared. When RGDS at 50 micrograms ml-1 was included in the incubation medium of skin explants, migration in fibronectin-coated dishes was almost totally blocked. In type I collagen-coated dishes, this concentration of RGDS also inhibited migration, but to a lesser degree than on fibronectin. With 250 micrograms ml-1 of RGES in the medium, the reverse was true. Here, migration on collagen was practically non-existent, while migration on fibronectin was affected only moderately. Collagen-mediated migration was sensitive to RGDS even when the peptide was added after migration on the coated substratum was well underway. At a coating concentration of 10 micrograms ml-1 CB3, a cyanogen bromide fragment of the collagen alpha 1(I) chain, which contains no RGD sequences, was as good a migration substratum as intact collagen applied at the same coating concentration. At lower concentrations intact collagen was somewhat better than equivalent concentrations of CB3. The presence of RGDS in the medium throughout an experiment inhibited migration in CB3-coated dishes in a manner similar to its effect in dishes coated with collagen. On both substrata there appeared to be a peptide-sensitive and a peptide-insensitive component to migration. The inhibitory effect of RGES on CB3-mediated migration was also similar to its effect in collagen-coated dishes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2005

Disruptions of the Arabidopsis Enoyl-CoA reductase gene reveal an essential role for very-long-chain fatty acid synthesis in cell expansion during plant morphogenesis.

In the absence of cell migration, plant architecture is largely determined by the direction and extent of cell expansion during development. In this report, we show that very-long-chain fatty acid (VLCFA) synthesis plays an essential role in cell expansion. The Arabidopsis thaliana eceriferum10 (cer10) mutants exhibit severe morphological abnormalities and reduced size of aerial organs. These mutants are disrupted in the At3g55360 gene, previously identified as a gene coding for enoyl-CoA reductase (ECR), an enzyme required for VLCFA synthesis. The absence of ECR activity results in a reduction of cuticular wax load and affects VLCFA composition of seed triacylglycerols and sphingolipids, demonstrating in planta that ECR is involved in all VLCFA elongation reactions in Arabidopsis. Epidermal and seed-specific silencing of ECR activity resulted in a reduction of cuticular wax load and the VLCFA content of seed triacylglycerols, respectively, with no effects on plant morphogenesis, suggesting that the developmental phenotypes arise from abnormal sphingolipid composition. Cellular analysis revealed aberrant endocytic membrane traffic and defective cell expansion underlying the morphological defects of cer10 mutants.

Arabidopsis↗

PubMed2000

A LIS1/NUDEL/cytoplasmic dynein heavy chain complex in the developing and adult nervous system.

Mutations in mammalian Lis1 (Pafah1b1) result in neuronal migration defects. Several lines of evidence suggest that LIS1 participates in pathways regulating microtubule function, but the molecular mechanisms are unknown. Here, we demonstrate that LIS1 directly interacts with the cytoplasmic dynein heavy chain (CDHC) and NUDEL, a murine homolog of the Aspergillus nidulans nuclear migration mutant NudE. LIS1 and NUDEL colocalize predominantly at the centrosome in early neuroblasts but redistribute to axons in association with retrograde dynein motor proteins. NUDEL is phosphorylated by Cdk5/p35, a complex essential for neuronal migration. NUDEL and LIS1 regulate the distribution of CDHC along microtubules, and establish a direct functional link between LIS1, NUDEL, and microtubule motors. These results suggest that LIS1 and NUDEL regulate CDHC activity during neuronal migration and axonal retrograde transport in a Cdk5/p35-dependent fashion.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗

PubMed1996

Novel form of p21(WAF1/CIP1/SDI1) protein in phorbol ester-induced G2/M arrest.

Cell cycle progression requires activation of different cyclin-dependent kinases (CDKs) which are positively regulated by cyclins and negatively regulated by CDK inhibitors. Growth inhibition of the Calu-1 lung carcinoma cells induced with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent activator of protein kinase C, is associated with G2/M arrest and induction of expression of a novel, faster-migrating form of p21(WAF1/CIP1/SDI1) (p21) protein, an inhibitor of cyclin-dependent kinases. This faster-migrating p21 protein was also expressed in TPA-treated A549 lung carcinoma cells which also exhibited G2/M arrest but not in TPA-treated U937 leukemia cells, which only expressed a slower-migrating form of p21 protein. However, reverse transcriptase-polymerase chain reaction and Southern analysis demonstrated no evidence of novel splice in TPA-treated Calu-1 cells. On the other hand, immunoblotting analysis demonstrated that the faster-migrating p21 protein could be detected only by peptide antibody directed against the N terminus but not the C terminus, suggestive of truncation of the latter or protein modification that results in the loss of the C-terminal epitope. Correlation of G2/M arrest with expression of the faster-migrating p21 protein suggests that this novel form of p21 protein may be a mediator of G2/M arrest and growth inhibition.

Cell Line↗

PubMed1998

Wide distribution of laminin-5 gamma 2 chain in basement membranes of various human tissues.

Laminin 5 (LN5), a heterotrimer of laminin alpha 3. beta 3. and gamma 2 chains. is a laminin isoform which strongly promotes adhesion. migration. and scattering of cells through binding to integrins alpha 3 beta 1, alpha 6 beta 1 and alpha 6 beta 4. To get an insight into the physiological functions of LN5, we prepared a mouse monoclonal antibody to human laminin gamma 2 chain and used it for immunohistochemical analysis of laminin gamma 2 chain in normal human tissues. The basement membranes of various epithelial tissues, such as the skin, lung, small intestine, stomach, kidney and prostate, were immunostained with the anti-laminin gamma 2 chain monoclonal antibody. In addition, the basement membrane of the surface germinal epithelium in the ovary was also positive for laminin gamma 2 chain. These results suggest general roles of LN5 in the anchorage of various types of epithelial cells to the underlying basement membrane and in the expression of their cellular functions. Moreover, deposition of laminin gamma 2 chain around small arteries and veins was observed in the thymus and spleen. This lymphatic organ-specific expression of vascular LN5 might provide a novel function of LN5 in immune responses.

Adult↗

PubMed1984

Analysis of surface mu-chain expression in human lymphoblastoid cell lines that do not produce light chains.

It has been suggested that light chains (LC) are necessary for the surface expression of mu heavy chains. Fluorescent antibody screening of 42 human lymphoblastoid cell lines transformed in our laboratory, however, disclosed four lines that expressed surface mu-chains without LC. The biosynthesis, glycosylation, and turnover of mu-chains in these cell lines was compared to mu-chain production in cell lines synthesizing both heavy chains and LC. LC production could not be detected in the mu +LC- cell lines by either surface or biosynthetic labeling. The mu-chains expressed on the surface of the LC- cells appeared as disulfide-linked dimers and migrated slightly faster on SDS-polyacrylamide gels (70 Kd) than did mu-chains from IgM monomers (H2 L2) (78 Kd) after reduction. Biosynthetic labeling in the presence of tunicamycin demonstrated that the smaller size of free mu heavy chains was due to incomplete glycosylation of these molecules and not to amino acid deletions. The mu-chains produced by mu + LC- cells lines were degraded faster than mu-chains from LC+ cell lines, but their rate of transit to the cell surface was identical in both cell types. Thus, although LC are necessary for the formation of an intact antigen-binding site, they are not involved in the synthesis or expression of membrane mu-chains.

Acetylglucosaminidase↗

PubMed1989

Identification and characterization of subpopulations of the free alpha-subunit that vary in their ability to combine with chorionic gonadotropin-beta.

The free (uncombined) alpha-subunit of hCG is secreted in excess over alpha beta dimer from both malignant and nonmalignant trophoblast cells and is secreted ectopically from a variety of other malignant cell types. The free alpha-subunits from various sources are distinguishable from those that combine because they migrate more heterogeneously and more slowly on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) than dimer alpha. We have previously identified three posttranslational modifications that may contribute to the altered mobility of the free alpha-subunit and to its inability to combine with the beta-subunit: 1) preferential phosphorylation of the free alpha-subunit, 2) O-glycosylation of free alpha, and 3) differences in the processing of the asparagine-linked oligosaccharides between the free and combinable forms. We have purified three populations of the alpha-subunit from the JAR choriocarcinoma cell line and from ChaGo, a bronchogenic carcinoma cell line that ectopically synthesizes only the alpha-subunit, in order to identify the posttranslational modifications that contribute to the altered mobility on SDS-PAGE. Fractionation of the oligosaccharides released from the alpha forms with peptide N-glycosidase has shown that the faster migrating alpha forms on SDS-PAGE have less completely processed oligosaccharide chains. Twenty-two to 25% of the JAR free alpha and 35-41% of the ChaGo alpha forms that migrate the fastest on SDS-PAGE recombine with beta in an in vitro recombination assay under conditions where 62% of the dimer alpha form recombines. In contrast, only 5-12% and 16-21% of the JAR free alpha and ChaGo alpha forms, respectively, that migrate the slowest on SDS-PAGE recombine with beta. The form of JAR free alpha least capable of combining with beta contains on O-linked glycan on Thr-39. This same site is a substrate for phosphorylation by JAR cells. However, most of ChaGo alpha fails to recombine with beta even though ChaGo alpha contains little O-linked carbohydrate. These results suggest that the larger asparagine-linked complex glycans on the slower migrating alpha forms are the major limiting factor for subunit combination. Although these modifications may not be rate limiting for combination in the rough endoplasmic reticulum, they may prevent dimerization of the free subunits later in the secretory pathway.

Cell Line↗

PubMed1998

Integrin alpha3beta1-mediated interaction with laminin-5 stimulates adhesion, migration and invasion of malignant glioma cells.

Gliomas, characterized by their progressively invasive phenotype, express integrin alpha3beta1 as a major receptor for the extracellular matrix both in vivo and in vitro. Since the integrin alpha3beta1 has been shown to be a specific receptor for laminin-5 (alpha3beta3gamma2), we examined the effects of purified human laminin-5 on adhesion, migration and invasion of human glioma cells. Among different types of laminin variants and other matrix proteins including fibronectin and vitronectin, laminin-5 was most potent in promoting adhesion and migration of different kinds of glioma cells. Laminin-5-mediated adhesion and migration were specifically inhibited by monoclonal antibodies against integrin alpha3 and beta1 chains, confirming the role of integrin alpha3beta1 as the major laminin-5 receptor. Invasion of the reconstituted basement membrane (i.e., Matrigel) by glioma cells was also selectively stimulated by laminin-5. Out results show that laminin-5 is the major extracellular stimulant for glioma cell adhesion, migration and invasion. The immunohistochemical distribution of laminin gamma2 chain, a laminin subunit unique to laminin-5, showed that it was expressed in the tumor parenchyma of human glioma tissues. Expression of laminin alpha3, beta3 and gamma2 chains in glioma tissues and in glioma cell lines was also demonstrated at the messenger RNA level by reverse transcription polymerase chain reaction. Our results, taken together, show that laminin-5 may be involved in the invasive phenotype of malignant gliomas both in vitro and in vivo.

Antigens, CD↗

PubMedDate not supplied

Assessment of T-cell receptor beta-chain diversity by heteroduplex analysis.

The aim of this work was to search for a simple and alternative approach to the currently used methodologies for the analysis of T-cell receptor repertoire diversity. To this end we studied whether the heteroduplex analysis could be adapted to study the clonality of the T-cell receptor beta chain (TCRBV). We therefore analyzed, by sequencing, the molecular characteristics of the V-D-J junctions of numerous TCRBV chains from a variety of patients and from normal individuals, and compared the results with those obtained with the heteroduplex analysis. The latter procedure involves the amplification of the target TCRBV chains and the denaturation and renaturation of the amplified product to permit the random association of the distinct DNA strands encoding the different junctional regions. Whereas amplified material from polyclonal lymphoid cells migrates on a polyacrylamide gel as a "smear" of bands composed of different-sized polyclonal PCR fragments, the mismatched chains derived from oligoclonal populations migrate as discrete "heteroduplexes" and can be separated from the matched "homoduplex" obtained from homogeneous clonal cells. Our results provide evidence demonstrating that heteroduplex analysis can successfully be applied to the analysis of T-cell clonality in a variety of samples and can be complementary or substitute for the standard approach of TCR cloning and multiple sequencing of junctional regions. Thus, the procedure should facilitate the implementation of the analysis of TCR in diagnostic routine and should find applications in numerous physiologic and pathologic conditions.

Amino Acid Sequence↗

PubMed1998

Assembly of the epithelial Na+ channel evaluated using sucrose gradient sedimentation analysis.

Three subunits, alpha, beta, and gamma, contribute to the formation of the epithelial Na+ channel. To investigate the oligomeric assembly of the channel complex, we used sucrose gradient sedimentation analysis to determine the sedimentation properties of individual subunits and heteromultimers comprised of multiple subunits. When the alpha subunit was expressed alone, it first formed an oligomeric complex with a sedimentation coefficient of 11 S, and then generated a higher order multimer of 25 S. In contrast, individual beta and gamma subunits predominately assembled into 11 S complexes. We obtained similar results with expression in cells and in vitro. When we co-expressed beta with alpha or with alpha plus gamma, the beta subunit assembled into a 25 S complex. Glycosylation of the alpha subunit was not required for assembly into a 25 S complex. We found that the alpha subunit formed intra-chain disulfide bonds. Although such bonds were not required to generate an oligomeric complex, under nonreducing conditions the alpha subunit formed a complex that migrated more homogeneously at 25 S. This suggests that intra-chain disulfide bonds may stabilize the complex. These data suggest that the epithelial Na+ channel subunits form high order oligomeric complexes and that the alpha subunit contains the information that facilitates such formation. Interestingly, the ability of the alpha, but not the beta or gamma, subunit to assemble into a 25 S homomeric complex correlates with the ability of these subunits to generate functional channels when expressed alone.

Animals↗

PubMed2005

Laminin-5 gamma 2 chain expression in cervical intraepithelial neoplasia and invasive cervical carcinoma.

BACKGROUND: To analyze the expression of laminin-5 gamma 2 chain, a protein which plays a major role in keratinocyte migration, in cervical intraepithelial neoplasia (CIN), and invasive cervical carcinoma associated with high-risk oncogenic human papillomaviruses (HR-HPVs). MATERIAL AND METHODS: The expression of laminin-5 gamma 2 chain protein has been analyzed by immunohistochemistry in 17 cases of low-grade squamous intraepithelial lesions (LSIL-CIN1), 35 high-grade squamous intraepithelial lesions (HSIL-CIN2/3), 18 microinvasive or invasive carcinomas, and three metastatic lymph nodes. All these lesions have been proved to contain HR-HPVs and were also positive for p16 protein which classically is overexpressed at all stages of cervical neoplasia and dysplasia linked with HR-HPVs. 20 cases of normal cervix served as controls. RESULTS: The expression of laminin-5 gamma 2 chain protein was observed in 100% of invasive or microinvasive carcinoma and in their related lymph node metastasis with an immunoreactivity located preferentially at the invasive front of the lesions. All the HSILs (100%) associated with invasive carcinoma were also positive. In contrast, in HSILs without associated invasive component, the expression of the protein has been found in only 34% of cases. In positive HSILs, laminin-5 gamma 2 protein was expressed in basal layers. In LSILs and normal epithelium, no expression of the protein was noted. CONCLUSIONS: We conclude the following: (i) the expression of laminin-5 gamma 2 is a late event in cervical carcinogenesis increasing with the grade of dysplastic lesions; (ii) laminin-5 gamma 2 expression facilitates the identification of invasive and microinvasive lesions which could be difficult to diagnose on the basis of routine stains; (iii) laminin-5 gamma 2 expression in HSILs could potentially identify those lesions with a more increased risk of tumor progression.

Biomarkers, Tumor↗

PubMed1990

Identification of an amino acid sequence from the laminin A chain that stimulates metastasis and collagenase IV production.

Tumor cells attach, degrade, and migrate through basement membranes as they metastasize. Laminin, a major glycoprotein of basement membranes, promotes the metastatic activity of tumor cells by stimulating the attachment and migration of the cells and their secretion of collagenase IV. We have identified a synthetic peptide of 19 amino acids (Cys-Ser-Arg-Ala-Arg-Lys-Gln-Ala-Ala-Ser-Ile-Lys-Val-Ala-Val-Ser-Ala-Asp -Arg) from the sequence of the A chain of laminin that increases experimental metastases of the lungs by murine melanoma cells. The peptide is active when injected either intravenously or intraperitoneally. The peptide increased collagenase IV activity, a key enzyme in the breakdown of basement membranes, to the same extent as laminin. This peptide represents an active site on laminin for promotion of the metastatic phenotype and generates a probe for studying the regulation of malignant activities.

Amino Acid Sequence↗

PubMed1995

Component C3 of hagfish complement has a unique structure: identification of native C3 and its degradation products.

A protein from hagfish serum that cross-reacted with the third component of hagfish complement (C3) was purified to homogeneity and its structural properties were compared with those of C3 which has a two-subunit chain structure (115 and 72 kDa). This protein (designated C3b), when purified from plasma, consisted of three disulfide-linked polypeptide chains (77, 72 and 30 kDa). On immunoelectrophoresis, purified C3b migrated more rapidly towards the anode than the beta mobility of C3. However, immunochemical analysis revealed that C3b, after the first step in its purification, consisted of two disulfide-linked polypeptide chains (105 and 72 kDa). Treatment of C3b with methylamine, prior to spectrophotometric titration of the free sulfhydryl groups, did not significantly affect the end-point of the titration, suggesting the absence of a thioester bond in this molecule. Analysis of the amino acid sequences of the amino-termini of the subunits of C3b revealed that 77 amino acid residues at the amino-terminus of the native alpha chain were missing from both the 77-kDa and the 105-kDa polypeptides from C3b. These results indicate that the C3b in this study was analogous to mammalian C3b. Furthermore, amino acid sequencing data indicated that most of the native C3 from hagfish serum has an irregular two-subunit (alpha+gamma and beta)-linked structure, as a result of one-sided processing of putative hagfish pro-C3 at the beta-alpha processing site exclusively. Moreover, it appears that only the molecular features of degenerated hagfish C3 (C3b) are altered during its purification to generate a three-chain structure.

Amino Acid Sequence↗

PubMed1993

Molecular mechanisms of neural crest cell attachment and migration on types I and IV collagen.

We have examined the mechanisms involved in the interaction of avian neural crest cells with collagen types I and IV (Col I and IV) during their adhesion and migration in vitro. For this purpose native Col IV was purified from chicken tissues, characterized biochemically and ultrastructurally. Purified chicken Col I and Col IV, and various proteolytic fragments of the collagens, were used in quantitative cell attachment and migration assays in conjunction with domain-specific collagen antibodies and antibodies to avian integrin subunits. Neural crest cells do not distinguish between different macromolecular arrangements of Col I during their initial attachment, but do so during their migration, showing a clear preference for polymeric Col I. Interaction with Col I is mediated by the alpha 1 beta 1 integrin, through binding to a segment of the alpha 1(I) chain composed of fragment CNBr3. Neural crest cell attachment and migration on Col IV involves recognition of conformation-dependent sites within the triple-helical region and the noncollagenous, carboxyl-terminal NC1 domain. This recognition requires integrity of inter- and intrachain disulfide linkages and correct folding of the molecule. Moreover, there also is evidence that interaction sites within the NC1 domain may be cryptic, being exposed during migration of the cells in the intact collagen as a result of the prolonged cell-substratum contact. In contrast to Col I, neural crest cell interaction with Col IV is mediated by beta 1-class integrins other than alpha 1 beta 1.

Animals↗

PubMed1984

Muscarinic cholinergic receptor of rat brain. Factors influencing migration in electrophoresis and gel filtration in sodium dodecyl sulphate.

The muscarinic cholinergic receptor present in synaptosomal membranes of rat brain was covalently labelled with the alkylating muscarinic antagonist, tritiated propylbenzilylcholine mustard. The labelled receptor was then solubilized in sodium deoxycholate and sodium dodecyl sulphate, and its migration in polyacrylamide gel electrophoresis and gel filtration in the presence of sodium dodecyl sulphate analysed. Provided both proteolysis and inter-chain disulphide bond formation were vigorously prevented, the receptor from rat forebrain (cerebral cortex plus caudate putamen) migrated, in sodium dodecyl sulphate/polyacrylamide gel electrophoresis, as a broad band of apparent Mr 66000-76000. Two dominantly labelled polypeptides, of apparent Mr 68000 and 73000, could be distinguished as the major components of this band. These multiple species seen in electrophoresis may reflect a structural diversity related to the different binding properties, and modes of action, of this receptor. In electrophoresis using discontinuous buffer systems the labelled receptor readily formed intermolecular disulphide bonds and so aggregated. In particular, if solubilized membranes were reduced with 2-mercaptoethanol, and reformation of disulphide bonds during electrophoresis not prevented, then formation of a dimeric species (apparent Mr 119000-128000) occurred. This probably explains previous reports in the literature of larger-Mr species seen in electrophoresis. During gel filtration, the receptor formed intra-chain disulphide bonds which produced conformational heterogeneity, leading to polydisperse migration. In addition, extensive proteolytic degradation of the receptor occurred due to a protease migrating slightly ahead of the receptor. Both effects were eliminated by alkylation of the solubilized membranes with iodoacetamide before gel filtration. Alkylated receptor migrated on Sephacryl S-300 in 0.5% sodium dodecyl sulphate with an equivalent Stokes' radius of 6.1 nm. This is identical to that of reduced ovalbumin, a molecule with an apparent Mr in gel electrophoresis of 43000. On a different gel matrix, TSK HW 55(S), the receptor migrated with a somewhat larger Stokes' radius, eluting just behind reduced bovine serum albumin (Stokes' radius 8.5 nm; apparent Mr in electrophoresis 67000). Thus the receptor appears to adsorb to the Sephacryl matrix, although even on the TSK gel the receptor eluted as a somewhat smaller protein than expected from its behaviour in gel electrophoresis. Solubilized, alkylated receptor, partly purified by gel filtration so that it was not degraded by endogenous proteases, was not cleaved by mild hydroxylamine treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1997

The alpha7beta1 integrin mediates adhesion and migration of skeletal myoblasts on laminin.

Many aspects of myogenesis are believed to be regulated by myoblast interactions with specific components of the extracellular matrix. For example, laminin has been found to promote adhesion, migration, and proliferation of mammalian myoblasts. Based on affinity chromatography, the alpha7beta1 integrin has been presumed to be the major receptor mediating myoblast interactions with laminin. We have prepared a monoclonal antibody, O26, that specifically reacts with both the X1 and the X2 extracellular splice variants of the alpha7 integrin chain. This antibody completely and selectively blocks adhesion and migration of rat L8E63 myoblasts on laminin-1, but not on fibronectin. In contrast, a polyclonal antibody to the fibronectin receptor, alpha5beta1 integrin, blocks myoblast adhesion on fibronectin, but not on laminin-1. The alpha7beta1 integrin also binds to a mixture of laminin-2 and laminin-4, the major laminin isoforms in developing and adult skeletal muscle, but O26 is a much less potent inhibitor of myoblast adhesion on the laminin-2/4 mixture than on laminin-1. Based on affinity chromatography, we suggest that this may be due to higher affinity binding of alpha7X1 to laminin-2/4 than to laminin-1.

Alternative Splicing↗

PubMed2006

Induction of human neutrophil chemotaxis by Candida albicans-derived beta-1,6-long glycoside side-chain-branched beta-glucan.

Polysaccharide beta-1,3-D-glucans (beta-glucans) are components of the cell wall of various fungi and show immunomodulatory activities. beta-Glucans have been reported to enhance neutrophil accumulation during pathogenic fungi-induced lung inflammation. Therefore, we examined whether beta-glucans themselves possess chemotactic activities for human neutrophils. Among several kinds of beta-glucans, beta-1,6-long glucosyl side-chain-branched beta-glucan, isolated from Candida albicans [Candida soluble beta-D-glucan (CSBG)], dose-dependently induced neutrophil migration in a Boyden chamber system. In contrast, 1,6-monoglucosyl-branched beta-glucans, such as Sparassis crispa-derived beta-glucan (SCG) and grifolan (GRN), which were derived from nonpathogenic fungi, hardly induced neutrophil migration. Moreover, CSBG-induced neutrophil migration was inhibited completely by liposomes containing neutral glycosphingolipid lactosylceramide (LacCer; Galbeta1-4Glc-ceramide) but not NeuAcalpha2-3Galbeta1-4Glcbeta1-1'-Cer ganglioside. Furthermore, binding experiments demonstrated that CSBG bound to glycosphingolipids (such as LacCer) with a terminal galactose residue; however, SCG and GRN (1,6-monoglucosyl-branched beta-glucans) did not bind to LacCer. It is important that a Src kinase inhibitor protein phosphatase 1, a phosphatidylinositol-3 kinase (PI-3K) inhibitor wortmannin, and a Galpha(i/o) inhibitor pertussis toxin inhibited neutrophil migration toward CSBG. Taken together, our results suggest that beta-1,6-long glucosyl side-chain-branched beta-glucan CSBG binds to LacCer and induces neutrophil migration through the activation of Src family kinase/PI-3K/heterotrimeric G-protein signal transduction pathways.

Binding Sites↗

PubMed2006

Chemokine CXCL12 induces binding of ferritin heavy chain to the chemokine receptor CXCR4, alters CXCR4 signaling, and induces phosphorylation and nuclear translocation of ferritin heavy chain.

Chemokine receptor-initiated signaling plays critical roles in cell differentiation, proliferation, and migration. However, the regulation of chemokine receptor signaling under physiological and pathological conditions is not fully understood. In the present study, we demonstrate that the CXC chemokine receptor 4 (CXCR4) formed a complex with ferritin heavy chain (FHC) in a ligand-dependent manner. Our in vitro binding assays revealed that purified FHC associated with both the glutathione S-transferase-conjugated N-terminal and C-terminal domains of CXCR4, thereby suggesting the presence of more than one FHC binding site in the protein sequence of CXCR4. Using confocal microscopy, we observed that stimulation with CXCL12, the receptor ligand, induced colocalization of the internalized CXCR4 with FHC into internal vesicles. Furthermore, after CXCL12 treatment, FHC underwent time-dependent nuclear translocation and phosphorylation at serine residues. By contrast, a mutant form of FHC in which serine 178 was replaced by alanine (S178A) failed to undergo phosphorylation, suggesting that serine 178 is the major phosphorylation site. Compared with the wild type FHC, the FHC-S178A mutant exhibited reduced association with CXCR4 and constitutive nuclear translocation. We also found that CXCR4-mediated extracellular signal-regulated kinase 1/2 (ERK1/2) activation and chemotaxis were inhibited by overexpression of wild type FHC but not FHC-S178A mutant, and were prolonged by FHC knockdown. In addition to CXCR4, other chemokine receptor-initiated signaling appeared to be similarly regulated by FHC, because CXCR2-mediated ERK1/2 activation was also inhibited by FHC overexpression and prolonged by FHC knockdown. Altogether, our data provide strong evidence for an important role of FHC in chemokine receptor signaling and receptor-mediated cell migration.

Active Transport, Cell Nucleus↗